Random mutagenesis of presenilin-1 identifies novel mutants exclusively generating long amyloid β-peptides

Familial Alzheimer disease-causing mutations in the presenilins increase production of longer pathogenic amyloid beta-peptides (A beta(42/43)) by altering gamma-secretase activity. The mechanism underlying this effect remains unknown, although it has been proposed that heteromeric macromolecular complexes containing presenilins mediate gamma-secretase cleavage of the amyloid beta-precursor protein. Using a random mutagenesis screen of presenilin-1 (PS1) for PS1 endoproteolysis-impairing mutations, we identified five unique mutants, including R278I-PS1 and L435H-PS1, that exclusively generated a high level of A beta(43), but did not support physiological PS1 endoproteolysis or A beta(40) generation. These mutants did not measurably alter the molecular size or subcellular localization of PS1 complexes. Pharmacological studies indicated that the up-regulation of activity for A beta(43) generation by these mutations was not further enhanced by the difluoroketone inhibitor DFK167 and was refractory to inhibition by sulindac sulfide. These results suggest that PS1 mutations can lead to a wide spectrum of changes in the activity and specificity of gamma-secretase and that the effects of PS1 mutations and gamma-secretase inhibitors on the specificity are mediated through a common mechanism.